Roger R. Fan, Leigh M. Howard, Marie R. Griffin, Kathryn M. Edwards,

Yuwei Zhu, John V. Williams, Jorge E. Vidal, Keith P. Klugman, Ana I. Gil,

Claudio F. Lanata, Carlos G. Grijalva

We examined nasopharyngeal pneumococcal colonization density patterns surrounding acute respiratory illnesses (ARI) in young children in Peru. Pneumococcal densities were dy-namic, gradually increasing leading up to an ARI, peaking during the ARI, and decreasing after the ARI. Rhinovirus co-infection was associated with higher pneumococcal densities.

Streptococcus pneumoniae commonly colonizes the nasopharynx of young children (1). Nasopharyngeal

colonization density is relevant for transmission of bacteria and pathogenesis of pneumococcal diseases (2). Few stud-ies have evaluated the longitudinal relationship between nasopharyngeal pneumococcal density and acute respira-tory illnesses (ARIs). We examined the evolution of na-sopharyngeal pneumococcal density surrounding ARIs in young children.

The StudyWe performed sequential cross-sectional assessments from a prospective cohort study of Andean children in Peru (3). Dur-ing 2009–2011, children <3 years of age from the District of San Marcos, Cajamarca, Peru, were assessed for ARIs dur-ing weekly household visits. The population was rural and had low incomes and limited access to healthcare (3,4). Use of 7-valent pneumococcal conjugate vaccine (PCV7) started in late 2009. Institutional review boards of Vanderbilt Uni-versity (Nashville, TN, USA) and the Instituto de Investiga-cion Nutricional (Lima, Peru) approved the study.

An ARI episode was defined as the length of time a child had cough or fever (5,6). If a child was ill during a household visit, we assessed for pneumonia or lower

respiratory tract infection using IMCI-WHO (Integrated Management of Childhood Illness–World Health Organi-zation) criteria (5,7). If the child had an ARI during the pre-ceding 7 days, we collected a nasal swab sample and tested it for respiratory viruses by reverse transcription PCR at Vanderbilt University (6,8–11). Nasopharyngeal swab samples were collected monthly without regard to ARI and tested at Emory University (Atlanta, GA, USA) by using quantitative PCR for pneumococcal density determina-tions. For this study, we used samples collected in 2009 and 2011, representing periods before and after routine PCV7 use (12) (online Technical Appendix, http://wwwnc.cdc.gov/EID/article/22/11/16-0902-Techapp1.pdf).

Nasopharyngeal samples were classified according to their collection time surrounding ARIs: peri-ARI periods included pre-ARI (8–14 or 1–7 days before an ARI) and post-ARI (1–7 or 8–14 days after an ARI). Samples outside these periods were considered non-ARI samples. We com-pared log-transformed pneumococcal nasopharyngeal den-sities of samples from ARI, peri-ARI, and non-ARI periods by using multivariable quantile regression with robust SEs and adjusting for relevant covariates.

In secondary analyses, we assessed the role of respi-ratory viruses on pneumococcal density in children with ARIs. Because detection of nonrhinovirus respiratory vi-ruses in nasal swabs was infrequent, we grouped samples into 4 distinct groups: rhinovirus only, rhinovirus and other viruses, other viruses only, and negative for any viruses.

We examined the role of pneumococcal acquisition on pneumococcal density using pneumococci-positive naso-pharyngeal samples from children who had a sample collect-ed within the preceding 60 days. Samples were categorized as 1) new colonization if the prior sample was negative, 2) serotype persistence if the prior sample was the same sero-type, and 3) serotype replacement if the prior sample was a different serotype. If either serotype was nontypeable or unknown, the pattern was considered undetermined.

We assessed 3,579 nasopharyngeal samples from 833 children: 450 (12.6%) were collected during ARIs, 956 (26.7%) during peri-ARI periods, and 2,173 (57.8%) dur-ing non-ARI periods. The median age was 1.39 years. The median duration for ARIs was 8 days (interquartile range [IQR] 5–13 days). According to IMCI-WHO criteria, 33 samples were associated with pneumonia or severe pneu-monia (13) (Table).

Nasopharyngeal Pneumococcal Density and Evolution of Acute Respiratory Illnesses in

Young Children, Peru, 2009–2011

1996 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No.11, November 2016

DISPATCHES

Author affiliations: Vanderbilt University, Nashville, Tennessee, USA (R.R. Fan, L.M. Howard, M.R. Griffin, K.M. Edwards, Y. Zhu, C.F. Lanata, C.G. Grijalva); University of Pittsburgh, Pittsburgh, Pennsylvania, USA (J.V. Williams); Emory University, Atlanta, Georgia, USA (J.E. Vidal, K.P. Klugman); Instituto de Investigacion Nutricional, Lima, Peru (A.I. Gil, C.F. Lanata)

DOI: http://dx.doi.org/10.3201/eid2211.160902

Pneumococcal Density and Acute Respiratory Illness

Overall, 36.7% of nasopharyngeal samples were from children who had received >2 PCV7 doses and were con-sidered vaccinated. Approximately 5.0% of samples were from children who had received aminopenicillins, cotri-moxazole, chloramphenicol, or furazolidone within the 7 days preceding sample collection.

Quantitative PCR detected S. pneumoniae in 68.9% of nasopharyngeal samples; 78.9% of ARI and 65.3% of non-ARI samples were positive (p = 0.06). Unadjusted log-transformed pneumococcal densities varied by ARI periods (online Technical Appendix).

Adjusted analyses showed that densities peaked during ARIs. In post hoc adjusted comparisons, densities were high-er during the 1–7 days pre-ARI (p<0.0001), ARI (p<0.0001), 1–7 days post-ARI (p<0.0001), and 8–14 days post-ARI (p = 0.007) than during the non-ARI period (Figure 1).

Of 450 ARI nasopharyngeal samples, 435 (97%) had corresponding nasal swab samples available for identifica-tion of respiratory viruses; 299 (68.7%) tested positive for at least 1 virus. Rhinovirus, which was detected in 44.6% (194/435) of samples, was the most common virus (online Technical Appendix). The median log-transformed pneu-mococcal densities of 299 virus-positive samples and 136 virus-negative samples were not significantly different (4.73 vs 3.94, respectively; adjusted p = 0.06).

During ARI, the median log-transformed pneumococ-cal densities varied among virus groups: virus-negative (3.94, IQR 0.00–5.67; n = 136), nonrhinovirus (4.49, IQR 3.12–5.48; n = 105), rhinovirus-only (4.91, IQR

3.43–6.23; n = 147), and rhinovirus detected with other viruses samples (5.03, IQR 3.28–6.53; n = 47). In mul-tivariable analyses, the only significant difference was between rhinovirus-only and virus-negative samples (p = 0.02) (Figure 2).

For the colonization patterns assessment, 2,479 (69.3%) nasopharyngeal samples had another sample col-lected <60 days before the current sample; the median time between samples was 28 days. The median log-transformed pneumococcal densities among samples that represented new colonizations (5.14, IQR 3.56–6.24; n = 411), serotype replacement (5.49, IQR 4.53–6.44; n = 322), and serotype persistence (5.79, IQR 4.82–6.47; n = 489) were compared. In multivariable analysis, serotype-replacement (p = 0.005) and serotype-persistence (p = 0.0003) samples had higher density than new colonization samples. The difference be-tween serotype replacement and serotype persistence was not significant (p = 0.2).

ConclusionsOur findings demonstrate a dynamic evolution of pneu-mococcal densities before, during, and after ARI episodes among young children. We observed a gradual increase in pneumococcal density leading up to an ARI episode, peak density during symptomatic ARI, and a decrease in density post-ARI to levels similar to those in baseline non-ARI periods.

Our observations of higher densities during ARI than non-ARI episodes align with those in studies from Vietnam

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No.11, November 2016 1997

Table. Demographic characteristics for children from whom nasopharyngeal swab samples were collected during different periods surrounding ARIs, Peru, 2009–2011*

Characteristic

Period of sample collection

Total, N = 3,579

Non-ARI, n = 2,173

Pre-ARI Current ARI,

n = 450

Post-ARI 8–14 days,

n = 211 1–7 days, n = 222

1–7 days, n = 332

8–14 days, n = 191

No. children† 765 186 189 320 262 172 833 Demographics Median age, y, at sample collection 1.45 1.36 1.37 1.23 1.25 1.37 1.39 Male 50.5 56.4 52.3 52.9 49.4 48.2 51.1 Attend daycare equivalent 7.8 7.1 8.6 4.2 6.6 5.2 7.1 Patient’s home Traditional stoves for cooking 63.1 73.5 63.5 61.1 68.7 62.8 64.0 Running water 24.7 17.5 17.6 24.0 18.7 27.2 23.3 Sewer or septic tank 21.1 18.5 18.5 20.4 22.3 22.5 20.9 Electricity 42.3 34.6 37.4 38.7 40.7 41.9 40.9 Season and year at sample collection Fall 2009 4.4 8.1 6.8 9.8 6.0 6.8 5.7 Winter 2009 19.3 25.1 27.5 30.0 23.2 25.7 22.2 Spring 2009 23.0 19.0 17.1 19.3 23.2 19.9 21.8 Fall 2011 24.6 25.1 24.3 21.6 22.6 25.1 24.1 Winter 2011 28.7 22.8 24.3 19.3 25.0 22.5 26.2 Altitude, m, of residence 1,976–2,321, quartile 1 25.4 26.5 23.9 23.8 24.7 26.7 25.1 2,322–2,644, quartile 2 25.1 23.2 24.3 23.8 25.6 26.7 24.9 2,645–2,861, quartile 3 24.6 19.9 25.7 29.3 24.7 25.7 25.0 2,862–3,803, quartile 4 24.9 30.3 26.1 23.1 25.0 20.9 24.9 *Data are %, except for no. children. ARIs, acute respiratory illnesses; non-ARI, a period outside the pre-ARI, current ARI, and post-ARI periods. †No. children who contributed samples during each period.

DISPATCHES

and South Africa (14,15) and complement those assess-ments by illustrating the dynamic evolution of pneumococ-cal densities and the role of virus co-infections and pneu-mococcal colonization patterns. Unlike other studies that focused on hospitalized children, our community-based study showed relatively modest variations in nasopharyn-geal pneumococcal density.

Rhinovirus detection was associated with increased pneumococcal density during ARI. Although we observed an even higher median pneumococcal density in samples co-infected with rhinovirus and other respiratory viruses, the number of observations was small and statistical power to demonstrate significant differences was limited.

Compared with new colonization in our study, serotype persistence and replacement were associated with higher pneumococcal density. Because many new colonizations might ultimately succumb to host mechanisms and fail to establish stable colonization (2), the observed lower densi-ties might reflect a decline of pneumococcal populations

as clearance evolved. Nevertheless, although statistically significant, the differences in density were relatively mod-est, and we cannot establish the precise time of coloniza-tion or clearance in our samples.

Our study has several limitations. ARI identification depended on the presence of cough or fever, which are subjective but widely used for routine ARI surveillance (5–7). Because our study used household-based rather than health facility–based surveillance, severe disease was infrequent, precluding detailed assessments of dis-ease severity. Due to small numbers, we could not study serotype-specific pneumococcal densities. In addition, because the study was conducted in rural communities of Peru, caution is warranted when extrapolating our find-ings to other settings.

Our findings demonstrated that, among young chil-dren, nasopharyngeal pneumococcal density started in-creasing before the onset of ARI symptoms, peaked during symptomatic ARI, and decreased after symptoms subsided.

1998 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No.11, November 2016

Figure 1. Estimated median pneumococcal densities with 95% CIs (vertical bars) by acute respiratory illness (ARI) period. Estimates derived from a quantile regression model that accounted for sex, age, daycare attendance, electricity, water supply, housing materials, kitchen type, smokers at home, vaccination, antimicrobial drug use, season, and altitude of residence. Asterisk indicates significantly different from ARI samples; dagger indicates significantly different from non-ARI samples.

Figure 2. Pneumococcal densities of current acute respiratory illness samples subdivided by reverse transcription PCR detection of respiratory viruses. Each circle represents a single bacterial density measurement. The median for the samples of each subgroup is represented by a gray horizontal line. Asterisk indicates significantly different from virus-negative samples.

Pneumococcal Density and Acute Respiratory Illness

Rhinovirus co-infection, serotype persistence, and serotype replacement were associated with increased nasopharyn-geal pneumococcal density. Nasopharyngeal pneumococ-cal density is dynamic surrounding ARI episodes and likely driven by complex virus–bacteria–host interactions.

AcknowledgmentsWe are indebted to the communities of San Marcos, Cajamarca, Peru, for participating in this study and to the field workers and field supervisors whose efforts in difficult geographic areas and harsh weather conditions enabled the conduct of this study. We also acknowledge the approval and continuous support of the Cajamarca Health Region authorities.

This work was funded by a Vanderbilt University Clinical and Translational Science Award (grant UL1 RR024975) from the National Institutes of Health; an investigator-initiated research grant from Pfizer (IIR WS1898786 [0887X1-4492] to C.G.G. and IIR WS2079099 to J.E.V.); the Thrasher Research Fund (grant 02832-9 to C.G.G.); and a Thrasher Early Career Award (to L.M.H.).

C.G.G. has served as a consultant to Pfizer in unrelated work. M.R.G. receives grant funding from MedImmune. K.M.E. receives grant funding from Novartis in unrelated work. J.V.W. serves on a Scientific Advisory Board for Quidel and an In-dependent Data Monitoring Committee for GlaxoSmithKline, neither related to the present work. C.F.L. serves as a Scientific Advisor to Takeda and GlaxoSmithKline in subjects not related to the present work. All authors have submitted the International Committee of Medical Journal Editors Form for Disclosure of Potential Conflicts of Interest.

Mr. Fan is a medical student at Vanderbilt University School of Medicine in Nashville, Tennessee. His research interests include the epidemiology of respiratory illnesses and host–pathogen interactions.

References 1. Simell B, Auranen K, Käyhty H, Goldblatt D, Dagan R,

O’Brien KL; Pneumococcal Carriage Group. The fundamental link between pneumococcal carriage and disease. Expert Rev Vaccines. 2012;11:841–55. http://dx.doi.org/10.1586/erv.12.53

2. Siegel SJ, Weiser JN. Mechanisms of bacterial colonization of the respiratory tract. Annu Rev Microbiol. 2015;69:425–44. http://dx.doi.org/10.1146/annurev-micro-091014-104209

3. Budge PJ, Griffin MR, Edwards KM, Williams JV, Verastegui H, Hartinger SM, et al.; RESPIRA-PERU Group. A household-based study of acute viral respiratory illnesses in Andean children. Pediatr Infect Dis J. 2014;33:443–7. http://dx.doi.org/10.1097/INF.0000000000000135

4. Grijalva CG, Griffin MR, Edwards KM, Williams JV, Gil AI, Verastegui H, et al. Cohort profile: the study of respiratory

pathogens in Andean children. Int J Epidemiol. 2014;43:1021–30. http://dx.doi.org/10.1093/ije/dyt065

5. Lanata CF, Rudan I, Boschi-Pinto C, Tomaskovic L, Cherian T, Weber M, et al. Methodological and quality issues in epidemiological studies of acute lower respiratory infections in children in develop-ing countries. Int J Epidemiol. 2004;33:1362–72. http://dx.doi.org/10.1093/ije/dyh229

6. Poehling KA, Edwards KM, Weinberg GA, Szilagyi P, Staat MA, Iwane MK, et al.; New Vaccine Surveillance Network. The underrecognized burden of influenza in young children. N Engl J Med. 2006;355:31–40. http://dx.doi.org/10.1056/NEJ-Moa054869

7. Gove S; the WHO Working Group on Guidelines for Integrated Management of the Sick Child. Integrated management of childhood illness by outpatient health workers: technical basis and overview. Bull World Health Organ. 1997;75 (Suppl 1):7–24.

8. Griffin MR, Walker FJ, Iwane MK, Weinberg GA, Staat MA, Erdman DD; New Vaccine Surveillance Network Study Group. Epidemiology of respiratory infections in young children: insights from the new vaccine surveillance network. Pediatr Infect Dis J. 2004;23(Suppl):S188–92. http://dx.doi.org/10.1097/01.inf.0000144660.53024.64

9. Kodani M, Yang G, Conklin LM, Travis TC, Whitney CG, Anderson LJ, et al. Application of TaqMan low-density arrays for simultaneous detection of multiple respiratory pathogens. J Clin Microbiol. 2011;49:2175–82. http://dx.doi.org/10.1128/JCM.02270-10

10. Klemenc J, Asad Ali S, Johnson M, Tollefson SJ, Talbot HK, Hartert TV, et al. Real-time reverse transcriptase PCR assay for improved detection of human metapneumovirus. J Clin Virol. 2012;54:371–5. http://dx.doi.org/10.1016/j.jcv.2012.05.005

11. Lu X, Holloway B, Dare RK, Kuypers J, Yagi S, Williams JV, et al. Real-time reverse transcription–PCR assay for comprehensive detection of human rhinoviruses. J Clin Microbiol. 2008;46:533–9. http://dx.doi.org/10.1128/JCM.01739-07

12. Hanke CR, Grijalva CG, Chochua S, Pletz MW, Hornberg C, Edwards KM, et al. Bacterial density, serotype distribution and antibiotic resistance of pneumococcal strains from the nasopharynx of Peruvian children before and after pneumococcal conjugate vaccine 7. Pediatr Infect Dis J. 2016;35:432–9. http://dx.doi.org/10.1097/INF.0000000000001030

13. World Health Organization. Revised WHO classification and treatment of pneumonia in children at health facilities: evidence summaries. Geneva: the Organization; 2014.

14. Vu HT, Yoshida LM, Suzuki M, Nguyen HA, Nguyen CD, Nguyen AT, et al. Association between nasopharyngeal load of Streptococcus pneumoniae, viral coinfection, and radiologically confirmed pneumonia in Vietnamese children. Pediatr Infect Dis J. 2011;30:11–8. http://dx.doi.org/10.1097/INF.0b013e3181f111a2

15. Wolter N, Tempia S, Cohen C, Madhi SA, Venter M, Moyes J, et al. High nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia. J Infect Dis. 2014;210:1649–57. http://dx.doi.org/10.1093/infdis/jiu326

Address for correspondence: Carlos G. Grijalva, Department of Health Policy, Vanderbilt University School of Medicine, 2600 Village at Vanderbilt, 1500 21st Ave, Nashville, TN, USA, 37212; email: Carlos.grijalva@vanderbilt.edu

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 22, No.11, November 2016 1999

Page 1 of 8

Article DOI: http://dx.doi.org/10.3201/eid2211.160902

Nasopharyngeal Pneumococcal Density and Evolution of Acute Respiratory

Illnesses in Young Children, Peru, 2009–2011

Technical Appendix

Expanded Description of Methods

Study Population

Our prospective cohort study (RESPIRA-PERU) (1,2) was designed to examine the

epidemiology and etiology of acute respiratory illness (ARI) in children <3 years old in the

District of San Marcos, Cajamarca, Peru. The local population is mostly low income, living in

rural communities with limited access to healthcare (1). The 7-valent pneumococcal conjugate

vaccine (PCV7) was initially introduced into the communities in late 2009, and uptake slowly

increased throughout the study period. This study was approved by the institutional review

boards of Vanderbilt University (Nashville, TN, USA) and the Instituto de Investigacion

Nutricional (Lima, Peru).

Weekly Household Visits

In the RESPIRA-PERU study, local field workers were trained to interview parents about

respiratory signs and symptoms of respiratory illness based on the Integrated Management of

Childhood Illness (IMCI-WHO) protocol (3,4). For this study, an ARI episode was defined as

the period of time a child had either cough or fever (5,6). If ill at the time of the weekly

household visit, the presence of IMCI-WHO pneumonia danger signs (inability to drink or

breastfeed, persistent vomiting, convulsions, lethargy, unconsciousness, stridor, severe

malnutrition) or signs of lower respiratory tract infection (tachypnea, audible wheezing, chest

retractions, grunting, nasal flaring, stridor, or cyanosis) was assessed (4,5).

Page 2 of 8

Respiratory Samples

Nasal swabs were collected for each ARI episode (through 7 days after symptom

resolution), and processed as previously described (6,7). Samples were shipped to Vanderbilt

University for detection of influenza viruses, respiratory syncytial virus, human

metapneumovirus, rhinovirus, adenovirus, and parainfluenza viruses by real-time reverse

transcription PCR (RT–PCR) (6,8–10). Monthly nasopharyngeal (NP) samples were collected

according to WHO guidelines (11,12), transported in 1 mL of skim milk-tryptone-glucose-

glycerine (STGG) medium and frozen in the media at 70°C. Analyses at Emory University

(Atlanta, GA< USA) included culture and DNA extraction from Streptococcus pneumoniae

isolates and NP specimens using a QIAamp DNA Mini Kit (QIAGEN, Valencia, CA).

Colonization density was determined using a targeted lytA gene real-time quantitative PCR

(qPCR)(13,14). Pneumococcal colonization density (CFU/mL) was quantified using purified

genomic DNA from S. pneumoniae reference strain TIGR4 and serially diluted 10-fold to

prepare standards (4 × 10° to 4 × 106 CFU) (14). Standards were run along with DNA from NP

samples in a CFX96 real-time PCR detection system (Bio-Rad, Hercules, CA) using the software

Bio-Rad CFX manager (14). Pneumococcal serotyping was performed by multiplex PCR

(13,15,16). We used samples collected in 2009 and 2011, representing the time periods before

and after routine use of PCV7 in the study communities, respectively (17).

ARI and Peri-ARI Periods

The duration of an ARI episode was defined as the number of days a child had either

cough or fever (5,6). We defined peri-ARI periods including 8–14 days pre-ARI, 1–7 days pre-

ARI, 1–7 days post-ARI and 8–14 days post-ARI. Samples collected outside the above

categories were classified as non-ARI. When a sample met criteria for >1 category because it

was collected between 2 ARIs, it was classified relative to the closest ARI.

Patterns of Pneumococcal Serotype Acquisition

A secondary analysis included all pneumococcal-positive samples in which there was a

previous NP sample within 60 days. When the previous sample was qPCR negative, the current

sample was classified as new colonization. When the previous sample was qPCR positive, the

current sample was classified as persistence if the serotype was the same, or as replacement if a

different serotype was detected. If either serotype was nontypeable or unknown, then the pattern

was considered undetermined. Unknown refers to those samples for which the serotyping

Page 3 of 8

reactions were inconclusive. Some samples that are lytA positive may contain insufficient

pneumococcal material for the multiplex PCR reactions to test positively for any serotype.

Statistical Analyses

We compared pneumococcal NP densities during ARI, peri-ARI, and non-ARI periods.

Bacterial densities were presented on a logarithmic scale. To retain the samples with zero

density, we applied a log10(x+1) transformation, where x represents the measured density. The

median density was chosen as the most appropriate measure of central tendency as it is more

robust to extreme values. Due to the persistent non-normal distribution of the transformed

values, we used multivariable quantile regression to investigate the relationship between the log-

transformed pneumococcal densities, periods of observation and relevant covariates. These

covariates included sex, age, daycare attendance, electricity, water supply, housing materials,

kitchen type, smokers at home, vaccination, antimicrobial drug use, season, and altitude. We

used a restricted cubic spline function to allow for nonlinear effects of age in the model. Since a

child could contribute >1 sample for our analyses, we accounted for this correlation of

observations by calculating robust SEs using the Huber-White Sandwich variance estimator. The

median pneumococcal densities for comparison groups were calculated using adjusted

predictions from the respective quantile regression models, while integrating the influence of

other model parameters. For post hoc comparisons of adjusted densities between the non-ARI

and the ARI periods, we conservatively applied a Bonferroni correction to the type I error (5

comparisons, and a corrected p<0.01 to define statistical significance).

We conducted secondary analyses to assess the role of respiratory viruses on

pneumococcal density, restricting the analysis to samples collected during ARI or post-ARI

periods. Given that rhinoviruses were commonly detected but other viruses were less frequently

detected, samples were classified into 4 mutually exclusive groups: 1) positive for rhinovirus

only, 2) positive for rhinovirus and other viruses, 3) positive for other viruses only; and 4)

negative for any viruses. Another secondary analysis examined the role of the previously

described patterns of pneumococcal serotype acquisition on pneumococcal density. For all

secondary analyses, we used multivariate quantile regression models to account for covariates.

Stata® version 14.0 was used for all statistical analyses (StataCorp, College Station, TX, USA).

Page 4 of 8

Expanded Description of Results

Summary of Enrollment and Surveillance in Parent RESPIRA-Peru Study

Enrollment and surveillance in the RESPIRA-Peru study have been described elsewhere

(1). In brief, during May 2009–September 2011, a total of 892 children were enrolled, 55,661

household visits were scheduled, 89% were executed (i.e., field workers reached their target

households), and 79% were successfully completed (i.e., information was collected during the

visit). A total of 4,655 nasal and 10,722 NP swabs were collected. Collection of NP swabs was

completed in 88% of scheduled collections.

Additional Details on Pneumococcal Density Assessments

The unadjusted log-transformed pneumococcal densities are shown for each period in

Technical Appendix Figure. In unadjusted analysis, the median NP density during the non-ARI

periods (3.71, IQR 0.00–5.59) was statistically lower than the 1–7 days pre-ARI (4.42, IQR

2.72–5.86; p<0.001), the ARI period (4.49, IQR 3.04–5.95; p<0.001), and 1–7 days post-ARI

(4.48, IQR 0.00–5.79; p = 0.002) periods.

In the multivariable quantile regression model that examined the association between

ARI study periods and pneumococcal density, season was significantly associated with median

pneumococcal density. Spring (October, November) 2009, Fall (May, June) 2011, and Winter

(July, August, September) 2011 were associated with elevated density compared to Fall 2009

and Winter 2009. Housing characteristics (including running water, electricity, and sewage

system), PCV7 vaccination, health facility visits, antimicrobial drug use and the presence of

smokers in the home did not have statistically significant associations with median

pneumococcal density.

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study of respiratory pathogens in Andean children. Int J Epidemiol. 2014;43:1021–30. PubMed

http://dx.doi.org/10.1093/ije/dyt065

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PERU Group. A household-based study of acute viral respiratory illnesses in Andean children.

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developing countries. Int J Epidemiol. 2004;33:1362–72. PubMed

http://dx.doi.org/10.1093/ije/dyh229

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Technical Appendix Table 1. Viral detections in nasal swab samples*

Virus No. Nasal swab samples, %

Rhinovirus 257 51.2 Adenovirus 84 16.7 Influenza 46 9.2 Parainfluenza 1–3 42 8.4 Respiratory syncytial virus 40 8.0 Human metapneumovirus 33 6.6 *Some samples had >1 virus detected.

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Technical Appendix Table 2. Pneumococcal serotype detections in NP swab samples*

Serotype No. NP swab samples, %

1 4 0.2 10A 87 4.3 10F/10C/33C 14 0.7 11A 102 5.0 11A/11D 53 2.6 12F 2 0.1 13 51 2.5 14 49 2.4 15A 38 1.9 15A/15F 22 1.1 15B 37 1.8 15B/15C 31 1.5 15C 38 1.9 16F 15 0.7 17F 26 1.3 18A 1 0.1 18A/18B/18C 8 0.4 18A/18B/18C/18F 8 0.4 18C 9 0.4 19A 61 3.0 19F 159 7.8 2 1 0.1 20 6 0.3 21 16 0.8 22A 3 0.2 22F 16 0.8 22F/22A 8 0.4 23A 25 1.2 23B 57 2.8 23F 138 6.8 24A/24B/24F 6 0.3 25F 1 0.1 28A 7 0.3 3 29 1.4 31 6 0.3 33A/33F/37 8 0.4 33B 7 0.3 33B/33D 2 0.1 33F 10 0.5 34 22 1.1 35A 16 0.8 35A/35C/42 18 0.9 35B 29 1.4 35C 1 0.1 35F 27 1.3 35F/47F 21 1.0 38/25F 7 0.3 39 3 0.2 4 16 0.8 5 2 0.1 6A 42 2.1 6A/6B 51 2.5 6A/6B/6C 69 3.4 6B 111 5.4 6C 127 6.2 6D 5 0.2 7B/7C/40 11 0.5 7C 27 1.3 7F 5 0.2 8 3 0.2 9A 3 0.2 9A/9V 13 0.6 9L/9N 10 0.5 9N 1 0.1 9V 12 0.6 NT 229 11.2 Total 2,042 100 *When serotyping reactions were unable to differentiate between >2 serotypes, the multiple serotypes are reported. NP, nasopharyngeal.

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Technical Appendix Figure. Pneumococcal densities for each acute respiratory illness (ARI) period.

Each circle represents a single bacterial density measurement. The median for the samples of each

period is represented by a red line. *Significantly different from ARI samples. †Significantly different from

non-ARI samples.